@misc{SchulzStegenJungetal., author = {Schulz, Christian and Stegen, Sarah and Jung, Friedrich and K{\"u}pper, Jan-Heiner}, title = {Mono-CYP CHO model : a recombinant Chinese hamster ovary cell platform for investigating CYP-specific tamoxifen metabolism}, series = {International journal of molecular sciences}, volume = {26}, journal = {International journal of molecular sciences}, number = {9}, editor = {Lamb, David and Syed, Khajamohiddin}, publisher = {MDPI}, address = {Basel}, issn = {1422-0067}, doi = {10.3390/ijms26093992}, pages = {1 -- 24}, abstract = {The metabolism of drugs and foreign substances in humans typically involves multiple enzymatic steps, particularly in phase-1 biotransformation in the liver, where various cytochrome P450 monooxygenases (CYPs) play crucial roles. This complexity can lead to a wide range of metabolites. Understanding the contributions of individual CYPs and their interactions within these intricate enzyme cascades can be challenging. We recently developed an in vitro biotransformation platform employing various Chinese Hamster Ovarian (CHO) cell clones. These clones express human cytochrome P450 oxidoreductase (CPR), and each is defined by a specific human CYP enzyme expression, thus exhibiting no detectable endogenous CYP enzyme activity (mono-CYP CHO platform). In this study, we investigated whether the mono-CYP CHO platform is a suitable tool for modeling complex drug metabolization reactions in vitro. Tamoxifen (TAM) was selected as a model substance due to its role as a prodrug widely used in breast cancer therapy, where its main active metabolite, endoxifen, arises from a two-step metabolism primarily involving the CYP system. Specifically, the combined activity of CYP3A4 and CYP2D6 is believed to be essential for efficient endoxifen production. However, the physiological metabolization pathway of TAM is more complex and interconnected, and the reasons for TAM's therapeutic success and variability among patients are not yet fully understood. Analogous to our recently introduced mono-CYP3A4 CHO cells, we generated a CHO cell line expressing human CPR and CYP2D6, including analysis of CYP2D6 expression and specific activity. Comparative studies on the metabolization of TAM were performed with both mono-CYP CHO models individually and in co-culture with intact cells as well as with isolated microsomes. Supernatants were analyzed by HPLC to calculate individual CYP activity for each metabolite. All the picked mono-CYP2D6 clones expressed similar CYP2D6 protein amounts but showed different enzyme activities. Mono-CYP2D6 clone 18 was selected as the most suitable for TAM metabolization based on microsomal activity assays. TAM conversion with mono-CYP2D6 and -3A4 clones, as well as the combination of both, resulted in the formation of the expected main metabolites. Mono-CYP2D6 cells and microsomes produced the highest detected amounts of 4-hydroxytamoxifen and endoxifen, along with N -desmethyltamoxifen and small amounts of N , N -didesmethyltamoxifen. N -desmethyltamoxifen was the only TAM metabolite detected in notable quantities in mono-CYP3A4, while 4-hydroxytamoxifen and endoxifen were present only in trace amounts. In CYP2D6/3A4 co-culture and equal mixtures of both CYP microsomes, all metabolites were detected at concentrations around 50\% of those in individual clones, indicating no significant synergistic effects. In conclusion, our mono-CYP CHO model confirmed the essential role of CYP2D6 in synthesizing the active TAM metabolite endoxifen and indicated that CYP2D6 is also involved in producing the by-metabolite N , N -didesmethyltamoxifen. The differences in metabolite spectra between the two mono-CYP models highlight the CYP specificity and sensitivity of our in vitro system.}, language = {en} } @misc{AckermannPeilQuarzetal., author = {Ackermann, G. and Peil, M. and Quarz, C. and Schmidt, A. and Halaczkiewicz, M. and Thomas, A. D. and Stegm{\"u}ller, S. and Richling, E. and Manolikakes, G. and Christmann, M. and K{\"u}pper, Jan-Heiner and Schrenk, D. and Fahrer, J.}, title = {Molecular dosimetry of estragole and 1′-hydroxyestragole-induced DNA adduct formation, clastogenicity and cytotoxicity in human liver cell models}, series = {Archives of toxicology}, volume = {99}, journal = {Archives of toxicology}, number = {9}, publisher = {Springer}, address = {Berlin ; Heidelberg}, issn = {0340-5761}, doi = {10.1007/s00204-025-04084-2}, pages = {3769 -- 3785}, abstract = {The phenylpropene estragole (ES) is found in essential oils of herbs and spices, such as bitter fennel and basil. Humans are exposed to ES through the diet and phytomedicines. After its absorption, ES undergoes metabolic activation by CYP1A2 and SULT1A1 in the liver, which can give rise to DNA adducts and hepatocarcinogenesis. Until now, quantitative genotoxicity data for ES in human liver cells are scarce, correlating DNA adduct levels with critical effects such as clastogenicity. Here, we used human HepG2 and HepG2-CYP1A2 cells as well as primary human hepatocytes (PHH) to study the genotoxic, clastogenic and cytotoxic potential of ES and its crucial metabolite 1′-hydroxyestragole (1′OH-ES). In addition, primary rat hepatocytes (PRH) were used for selected endpoints. Treatment of HepG2-CYP1A2 cells with ES (0-2 mM) led to the concentration-dependent formation of E3′- N 2 -dG adducts. Apart from a moderate γH2AX induction, neither p53 accumulation nor cytotoxicity was observed. However, clastogenicity was demonstrated at ES concentrations ≥ 1 mM. Incubation of HepG2 cells with 1′OH-ES (0—35 µM) led to 10-50-fold higher E3′- N 2 -dG adduct levels compared to equimolar ES concentrations. Furthermore, 1′OH-ES caused γH2AX formation, p53 accumulation and cytotoxicity, which was confirmed in PHH. In agreement, 1′OH-ES induced clastogenicity at concentrations ≥ 25 µM. Molecular dosimetry revealed that a certain E3′- N 2 -dG adduct level is required to trigger clastogenicity and cytotoxicity. This was confirmed by Benchmark Concentration (BMC) modelling, showing that the BMC for clastogenicity is 12-17-fold higher than the respective BMC for DNA adduct formation. Our data indicate that a threshold level of DNA adducts is required, both in rat and human liver cells, to trigger markers of clastogenicity. These levels are unlikely to be reached in humans following chronic ES exposure through phytomedicines or the diet.}, language = {en} } @misc{NeruschSchichtHerzogetal., author = {Nerusch, Julia and Schicht, Gerda and Herzog, Natalie and K{\"u}pper, Jan-Heiner and Seehofer, Daniel and Damm, Georg}, title = {Investigation and distinction of energy metabolism in proliferating hepatocytes and hepatocellular carcinoma cells}, series = {Cells}, volume = {14}, journal = {Cells}, number = {16}, publisher = {MDPI}, address = {Basel}, issn = {2073-4409}, doi = {10.3390/cells14161254}, pages = {1 -- 26}, abstract = {Metabolic rewiring is a hallmark of both hepatic regeneration and malignant transformation, complicating the identification of cancer-specific traits. This study aimed to distinguish the metabolic profiles of proliferating hepatocytes and hepatocellular carcinoma (HCC) cells through integrated analyses of mRNA and protein expression, along with functional characterization. We compared non-malignant Upcyte® hepatocytes (HepaFH3) cultured under proliferative and confluent conditions with primary human hepatocytes, primary human hepatoma cells, and hepatoma cell lines. Proliferating HepaFH3 cells exhibited features of metabolic reprogramming, including elevated glycolysis, increased HIF1A expression, and ketone body accumulation, while maintaining low c-MYC expression and reduced BDH1 levels, distinguishing them from malignant models. In contrast, HCC cells showed upregulation of HK2, c-MYC, and BDH1, reflecting a shift toward aggressive glycolytic and ketolytic metabolism. Functional assays supported the transcript and protein expression data, demonstrating increased glucose uptake, elevated lactate secretion, and reduced glycogen storage in both proliferating and malignant cells. These findings reveal that cancer-like metabolic changes also occur during hepatic regeneration, limiting the diagnostic utility of individual metabolic markers. HepaFH3 cells thus provide a physiologically relevant in vitro model to study regeneration-associated metabolic adaptation and may offer insights that contribute to distinguishing regenerative from malignant processes. Our findings highlight the potential of integrated metabolic profiling in differentiating proliferation from tumorigenesis.}, language = {en} } @misc{CarlssonHerzogFelskeetal., author = {Carlsson, Max J. and Herzog, Natalie and Felske, Christina and Ackermann, Gabriel and Regier, Alexander and Wittmann, Simon and Fern{\´a}ndez Cereijo, Ra{\´u}l and Sturla, Shana J. and K{\"u}pper, Jan-Heiner and Fahrer, J{\"o}rg}, title = {The DNA repair protein MGMT protects against the genotoxicity of N-nitrosodimethylamine, but Not N-nitrosodiethanolamine and N-nitrosomethylaniline, in human HepG2 liver cells with CYP2E1 expression}, series = {Chemical research in toxicology}, volume = {38}, journal = {Chemical research in toxicology}, number = {6}, publisher = {American Chemical Society (ACS)}, address = {New York, NY}, issn = {0893-228X}, doi = {10.1021/acs.chemrestox.5c00133}, pages = {1134 -- 1146}, abstract = {N-nitrosamines are genotoxic contaminants that occur in the diet, consumer products, and the environment. More recently, N-nitrosamines were also detected as drug impurities. After uptake, N-nitrosamines undergo metabolic activation by cytochrome P450 monooxygenases (CYPs), resulting in DNA damage and tumor formation. In this study, the genotoxicity and cytotoxicity of three N-nitrosamines with structurally distinct substituents, N-nitrosodimethylamine (NDMA), N-nitrosodiethanolamine (NDELA) and N-nitrosomethylaniline (NMA), were analyzed in human HepG2 liver cell models proficient or deficient in CYP2E1 biotransformation. Furthermore, the impact of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) was investigated. The novel genetically engineered HepG2-CYP2E1 cell line strongly expressed CYP2E1, which was not detectable in wildtype (WT) HepG2 cells. We then confirmed that the CYP2E1 substrate NDMA caused O6-methyldesoxyguanosine adducts and DNA strand breaks in a CYP2E1-dependent manner, leading to cytotoxicity. By the same approach, we demonstrated that NDELA induced DNA strand breaks in HepG2-CYP2E1 cells, whereas no effect was observed for NMA. However, NMA was revealed to cause DNA cross-links. Furthermore, both NDELA and NMA were cytotoxic in HepG2-CYP2E1 cells, but not in WT cells. Subsequently, the pharmacological MGMT inhibitor O6-benzylguanine was used to deplete MGMT in both HepG2 cell models. MGMT inhibition clearly increased DNA strand break levels due to NDMA exposure, whereas DNA strand break formation by NDELA and NMA were not affected by inhibiting MGMT. In line with these findings, the clastogenic effects of NDMA were potentiated in the absence of MGMT. In contrast to that, NDELA- and NMA-induced clastogenicity was not influenced by MGMT inhibition. Taken together, our study revealed that all three structurally diverse N-nitrosamines are cytotoxic and clastogenic in a CYP2E1-dependent manner, while only NDMA and NDELA caused DNA strand breaks. Furthermore, we demonstrated for the first time that DNA repair by MGMT does not confer protection against NDELA and NMA-triggered DNA strand break induction and clastogenicity.}, language = {en} } @misc{BrauneJungKuepperetal., author = {Braune, Steffen and Jung, Conrad G. H. and K{\"u}pper, Jan-Heiner and Jung, Friedrich}, title = {Arthrospira platensis as protein-rich source for human nutrition}, series = {Life}, volume = {15}, journal = {Life}, number = {12}, publisher = {MDPI}, address = {Basel}, issn = {2075-1729}, doi = {10.3390/life15121789}, pages = {1 -- 15}, abstract = {The continuing growth of the world's population, combined with climate change, poses a growing challenge to ensuring food security in the 21st century. Animal protein, e.g., from beef, is a particularly rich source of protein, but there is by no means enough arable land on earth to transfer the animal meat-rich nutritional style of the early industrialized countries to the global South. A hitherto largely neglected option for the production of proteins is the cultivation of microalgae and cyanobacteria, which already have a long history of use as a human or animal food for their nutritional and environmental merits. In particular, Limnospira platensis (Spirulina and formerly Arthrospira platensis)—a filamentous cyanobacterium—is considered the "food of the future" since it is a viable source of vegan protein. In this manuscript, we review the scientific literature as well as national and intergovernmental agency statements regarding the quality and quantity of AP-based proteins. The content of AP protein is d}, language = {en} }